Structure of the Murine TRAF1 Gene Ian F
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Molecular Immunology 36 (1999) 611±617 www.elsevier.com/locate/molimm Structure of the murine TRAF1 gene Ian F. Dunn, Raif S. Geha, Erdyni N. Tsitsikov* Division of Immunology, Children's Hospital and Department of Pediatrics, Harvard Medical School, Ender 8th, 300 Longwood Avenue, Boston, MA 02115, USA Received 10 March 1999; accepted 7 April 1999 Abstract We have cloned, characterized and sequenced the murine TNF Receptor Associated Factor 1 (TRAF1) gene. Restriction mapping and Southern blotting analysis revealed that the TRAF1 gene comprises 10 exons and 9 intervening introns and spreads over 18 kb of genomic DNA. 5'-RACE analysis of the TRAF1 transcript using mRNA from activated spleen B cells revealed several transcription start sites between positions 42 to +4 relative to the 5'end of the murine TRAF1 cDNA sequence. We also isolated and sequenced the 5'-upstream promoter region, which lacks TATA-like and CAAT-like sites but contains GC-rich sequences. Taken together, these results suggest that the TRAF1 gene promoter is a member of the class of Sp-1-dependent promoters. Near the transcription initiation start site we identi®ed three identical decanucleotide repeats (CCAGCCCAGC) which may play a role in the transcriptional regulation of TRAF1 expression. In addition we show that TRAF1 mRNA is not expressed in non-stimulated lymphocytes but can be induced upon activation with dierent stimuli, including anti-CD3, anti-IgM, anti-CD40 antibodies, LPS, or a combination of phorbol-12-myristate-13-acetate and ionomycin. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: TRAF1; Gene structure; Promoter sequences 1. Introduction others. All of these receptors are devoid of intristic catalytic activity. Some of them, like TNFR-I and Members of the tumor necrosis factor alpha (TNF) CD95/Fas contain a death domain and bind to the superfamily are pro-in¯ammatory cytokines involved recently identi®ed signal transduction proteins like in a variety of physiological and pathological con- TRADD, FADD/MORT-1 and RIP. Other members ditions. TNF superfamily members include TNF, lym- of the TNFR superfamily, which lack a death domain photoxins a and b, CD30L, CD40L, CD70, CD95/ and include TNFR-II, CD40 and CD30, associate with Fas, 4-1BBL, and others. They interact with the mem- members of the TRAF (TNF Receptor Associated bers of TNF receptor (TNFR) superfamily and play Factor) family of intracellular signal transduction mol- important roles in a wide array of biological processes ecules (Arch et al., 1998). including the acute phase response, cell growth and TRAF molecules were ®rst described due to their apoptosis, in¯amation and lymphocyte activation ability to bind to TNF-RII (Rothe et al., 1994). Six (Baker and Reddy, 1996). The TNFR superfamily TRAF molecules have been identi®ed to date (Cao et includes the following members: TNF-RI, TNF-RII, al., 1996; Cheng et al., 1995; Hu et al., 1994; Ishida et LT-R, CD27, CD30, CD40, CD95, OX-40, 4-1BB, and al., 1996a, b; Mosialos et al., 1995; Nakano et al., 1996; Regnier et al., 1995; Sato et al., 1995). Individual TNFR family members may associate with one or several TRAFs, which in turn could result in a * Corresponding author. Tel.: +1-617-735-8205; fax: +1-617-735- wide spectrum of divergent physiological functions. In 4174. E-mail address: [email protected] (E.N. general, TRAFs are characterized by the presence of Tsitsikov) an N-terminal RING ®nger, several zinc ®ngers and a 0161-5890/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S0161-5890(99)00075-9 612 I.F. Dunn et al. / Molecular Immunology 36 (1999) 611±617 C-terminal TRAF domain. The eector function of ducts that function cooperatively at the earliest check- TRAF2 requires its ®nger domains whereas the TRAF point to suppress TNFa-mediated apoptosis. Thus, domain is important for hetero- and homotypic inter- TRAF1 is one of the key regulators of the cellular actions with receptors, other TRAF proteins, or other stress response. downstream signal transducers. It was shown in vitro To gain further insight into the TRAF1 function that TRAF2 is required for JNK/SAPK and NF-kB and to understand the molecular mechanisms respon- activation induced by two TNFRs, CD40, CD27, sible for the TRAF1 expression, we have cloned and TRANCE-R, 4-1BB and CD30 (Akiba et al., 1998; characterized the murine TRAF1 gene. Arch and Thompson, 1998; Gedrich et al., 1996; Horie et al., 1998; Jang et al., 1998; Lee et al., 1997; Natoli et al., 1997; Pullen et al., 1998; Rothe et al., 1995; 2. Materials and methods Tsitsikov et al., 1997; Wong et al., 1998; Yamamoto et al., 1998). Gene targeting experiments demonstrated 2.1. Cloning of the mTRAF1 cDNA the crucial importance of the TRAF2 and TRAF3 proteins in cell signaling. Although TRAF2 null mice To obtain the mTRAF1 cDNA we screened the appear normal at birth, then become progressively mouse spleen cDNA Lambda ZAPII library runted and die prematurely. Examination of the (Stratagene) with a 300 bp fragment of the hTRAF1 TRAF2-de®cient cells revealed a severe reduction in described earlier (Tsitsikov et al., 1997). This fragment TNF-mediated JNK/SAPK activation but a surpris- corresponds to the coding region of the hTRAF1 ingly mild eect on NF-kB activation (Yeh et al., cDNA right 5'-upstream of HindIII site. After screen- 1997). Mice lacking TRAF3 exhibited a profound fail- ing of 0.5 Â 106 phage plaques we isolated six positive ure in homeostasis of the hematopoetic system and clones of dierent sizes. other organ systems, and die by 10 days of age (Xu et al., 1996). 2.2. Cloning and characterization of the mTRAF1 gene TRAF1 is a unique member of the TRAF family. It does not have a RING ®nger domain and contains a The mTRAF1 gene was isolated by screening of the single zinc ®nger, N-TRAF and C-TRAF domains. Lambda FIXII library (Stratagene) which was made Originally, the murine TRAF1 was cloned by bio- from the liver of strain 129/Sv female mice. We used a chemical characterization of signal transducers associ- 750 bp piece of the murine cDNA located upstream of ated with the cytoplasmic domain TNF-RII. It was the HindII site as a probe for screening of the genomic puri®ed from the lysate of the murine interleukin-2- library. Screening of approximately 1 Â 106 plaques dependent cytotoxic T cell line by immunoprecipitation using mTRAF1 cDNA probe yielded 14 dierent posi- with human TNF-RII (Rothe et al., 1994). Human tive phages, which were used for further studies. TRAF1 (EB16) was cloned by association with Restriction mapping was carried out by partial diges- Epstein±Barr virus latent infection membrane protein tion and Southern blotting hybridization with T3 and 1 (LMP1) from EBV-transformed lymphoblastoid cell T7 oligonucleotide probes as suggested by manufac- line by the yeast two-hybrid screen (Mosialos et al., turer. After alignment of individual clones, appropriate 1995). We and others have also cloned TRAF1 in ad- DNA fragments were subcloned into pBluescript II KS dition to TRAF2 by yeast two-hybrid screening with (+) (Stratagene). Exons were localized by Southern the cytoplasmic domain of CD30 (Aizawa et al., 1997; blotting hybridization using synthetic oligonucleotides Ansieau et al., 1996; Boucher et al., 1997; Duckett et from dierent parts of the TRAF1 cDNA and exon/ al., 1997; Gedrich et al., 1996; Lee et al., 1996a; intron junctions were sequenced. The sequencing reac- Tsitsikov et al., 1997). Ultimately, it was shown that tions were performed using Sequenase 2.0 kit TRAF1 can be recruited to a variety of distinct mem- (Amersham) or by automatic sequencing in MRRC bers of the TNFR superfamily, including TNF-RII, DNA Sequencing Core facility at Children's Hospital, CD30, 4-1BB, OX-40, HVEM/ATAR, and TRANCE- Boston, MA. The promoter sequence and consensus R. Little is known about TRAF1 function except that nucleotide motif analysis were performed using the when overexpressed in transgenic mice TRAF1 plays GENETYX-MAC software (Software Development, an inhibitory role in antigen-induced apoptosis of Tokyo, Japan). CD8+T lymphocytes (Speiser et al., 1997). A biologi- cal role for TRAF1 as a regulator of apoptotic signals 2.3. Northern blotting analysis has been suggested (Lee et al., 1996b). Recently, TRAF1, TRAF2, and the inhibitor-of-apoptosis pro- Anti-CD3 (2C11) and anti-CD40 (HM40-3) were teins (IAP) were identi®ed as gene targets of NF-kB- purchased from Pharmingen. Anti-mouse IgM (mu dependent transcriptional activity (Wang et al., 1998). chain) F(ab')2 fragments were purchased from TRAF1 was in a group of NF-kB-dependent gene pro- Rockland Inc. LPS (Serotype 0111:B4) and PMA were I.F. Dunn et al. / Molecular Immunology 36 (1999) 611±617 613 Fig. 1. Genomic organization and restriction map of the murine TRAF1 gene (A). The 10 exons are represented by the closed boxes. Schematic representation of the murine and human TRAF1 genes in comparison with protein structure (B). In the protein, TRAF1 domains are represented by wider boxes and are indicated as zinc ®nger, N-TRAF and C-TRAF, respectively. In the genes, boxes represent exons and connecting lines represent introns. The coding sequences is represented by shaded areas. The hatched areas indicate untranslated sequences. purchased from Sigma. Ionomycin (Io) was purchased CTC GTTTTCATC. PCR products were subcloned from Calbiochem. Splenocytes were prepared from the into pCRII vector (Invitrogen) and sequenced. spleen of 2 months old C57BL/6 mice. Isolated cells were maintained in RPMI1640 medium supplemented with 10% FCS and 50 mM 2-mercaptoethanol and 3. Results and discussion activated overnight with dierent stimuli including anti-CD3 (2 mg/ml), anti-IgM (5 mg/ml), LPS (1 mg/ 3.1.